H. Vietsch

Antagonists of the Mannose Receptor and the LDL Receptor–Related Protein Dramatically Delay the Clearance of Tissue Plasminogen Activator

BACKGROUND Clinical application of tissue plasminogen activator (TPA) as a fibrinolytic agent is complicated by its rapid clearance from the bloodstream, which is caused by TPA liver uptake. The mannose receptor on endothelial liver cells and the LDL receptor-related protein (LRP) on parenchymal liver cells were reported to contribute to liver uptake. METHODS AND RESULTS In this study, we addressed whether TPA clearance can be delayed by inhibiting receptor-mediated endocytosis of TPA. A series of cluster mannosides was synthesized, and their affinity for the mannose receptor was determined. A cluster mannoside carrying six mannose groups (M6L5) displayed a subnanomolar affinity for the mannose receptor (Ki = 0.41 +/- 0.09 nmol/L). Preinjection of M6L5 (1.2 mg/kg) reduced the clearance of 125I-TPA in rats by 60% because of specific inhibition of the endothelial cell uptake. The low toxicity of M6L5, combined with its accessible synthesis and high specificity for the mannose receptor, makes it a promising agent to improve the pharmacokinetics of TPA. Blockade of LRP by 39-kD receptor-associated protein (GST-RAP) also inhibited TPA clearance by 60%. Finally, combined preinjection of M6L5 and GST-RAP almost completely abolished reduced liver uptake of TPA and delayed its clearance by a factor of 10. CONCLUSIONS It can be concluded that (1) the mannose receptor and LRP appear to be the sole major receptors responsible for TPA clearance and (2) therapeutic levels of TPA can be maintained for a prolonged time span by coadministration of the aforementioned receptor antagonists.

Effect of skin barrier competence on SLS and water-induced IL-1α expression

Abstract: For screening of a potential irritant it is essential that an early marker for irritation should be chosen which could be detected before the physiological signs of irritation occur. Interleukin 1 alpha (IL‐1α) is widely accepted as such a marker in both in vivo and in vitro test systems. In this study, we have determined the mRNA levels of IL‐1α in the epidermis after topical application of sodium dodecyl sulphate (SLS) in both a commercially available epidermal kit (EpiDerm) and in excised skin. Furthermore, we have determined the effect of water, the vehicle for SLS, on IL‐1α mRNA levels. Topical application of water to excised skin increases IL‐1α mRNA levels sixfold in the epidermis whereas topical application of water to EpiDerm cultures did not alter IL‐1α mRNA levels. This is explained by the finding that EpiDerm cultures have a sub‐optimal barrier function when compared with excised skin – topical application of SLS was clearly toxic at much lower concentrations in EpiDerm cultures (0.2% SLS) than in excised skin (5% SLS). Also caffeine penetration was 10‐fold higher through EpiDerm cultures than through the excised skin. Therefore, incubation of control EpiDerm cultures at 100% humidity effectively mimics topical exposure to water. An additional increase in IL‐1α mRNA levels observed between topical application of water and SLS is similar (about threefold) in both experimental systems. In conclusion, in vitro reconstructed epidermis models, such as EpiDerm, can be used as a predictive model for irritancy screening. However, great care should be taken when interpreting the results due to the fact that EpiDerm cultures do not have a competent barrier function and therefore lower irritant concentrations are required than in in vivo or ex vivo studies in order to induce cytotoxic effects. Furthermore, the irritant effects of the vehicle should not be neglected. Our results show clearly that the topical application of water to excised skin results in increased levels of IL‐1α mRNA in the epidermis. This is a cytokine that is widely used as an early marker for skin irritation.

[23] Targeted delivery of antisense oligonucleotides to parenchymal liver cells in vivo

Publisher Summary Oligodeoxynucleotides (ODNs) inhibit gene expression at various levels both in vitro and in vivo. In vivo , the efficacy of ODN-induced regulation of genes in specific cell types may be suboptimal owing to poor accumulation of ODNs in these cells. In addition, untimely elimination of ODNs through renal clearance, degradation, and scavenger receptor-mediated uptake may further impair their therapeutic activity. These hurdles can be at least partly overcome by targeted delivery of the ODNs to the desired site of action. As short (and in particular uncharged) oligothymidinylates are poor substrates for hepatic scavenger receptors that are responsible for the rapid elimination of ODNs by cells of the reticuloendothelial system, the results leave unanswered whether longer, charged, and miscellaneous ODN sequences can also be redirected to the aimed target cell in vivo. In this respect, it is crucial to analyze the tissue distribution and identify the cellular uptake sites within the liver, using full-length antisense sequences. In the study described in the chapter, in vivo evidence is provided that untimely elimination of a miscellaneous 20-mer ODN by the preceding scavenger pathways can be circumvented and, concomitantly, accumulation by parenchymal liver cells can be enhanced after derivatization with a small-sized synthetic galactoside with high affinity for the asialoglycoprotein receptor.

Cholesterol Derivative of a New Triantennary Cluster Galactoside Lowers Serum Cholesterol Levels and Enhances Secretion of Bile Acids in the Rat

BACKGROUND Previous studies have demonstrated that cholesterol-derivatized galactosides exert a hypocholesterolemic effect by inducing hepatic uptake of atherogenic lipoproteins by means of galactose-recognizing receptors in the liver. However, a prolonged infusion of high concentrations of these compounds was required for this effect, possibly because of low affinity for the galactose-recognizing asialoglycoprotein receptor on the parenchymal liver cell. METHODS AND RESULTS We have designed a new series of triantennary galactosides to optimize the affinity and specificity for this receptor. The affinity of a triantennary galactoside for the asialoglycoprotein receptor appeared to be dramatically enhanced by proper spacing of the three terminal galactose groups. In rats, a single injection of N-[tris-O-(3,6,9-trioxaundecanyl-beta-D-galacto- pyranosyl)methoxymethyl]methyl-N alpha-[1-(6-(5-cholesten-3 beta- yloxy)glycyl)adipyl]glycinamide [TG(20A)C], the cholesterol derivative of the most selective galactoside, causes a dose-dependent decrease of < or = 45% in the serum cholesterol concentration (P < .001). This decrease is mainly attributed to a decrease in the level of serum HDL (P = .0066) and, to a lesser extent, serum LDL (P = .036). In addition, TG(20A)C strongly enhances the bile-acid secretion in rats during the first 2 hours after administration, which indicates that TG(20A)C-induced clearance of cholesterol from the bloodstream is efficiently coupled to hepatic bile-acid secretion. CONCLUSIONS We conclude that TG(20A)C efficiently directs lipoproteins that contain cholesterol to the liver at a 30-fold-lower concentration than previously developed cholesterol-derived cluster galactosides. This newly developed approach to lower cholesterol levels may prove valuable for familial hypercholesterolemic patients or those with familial defective apolipoprotein B-100 who do not respond or who respond insufficiently, respectively, to conventional therapies.

Induction of Hepatic Uptake of Lipoprotein(a) by Cholesterol-Derivatized Cluster Galactosides

We have previously developed triantennary galactosides [TG(4A)C and TG(20A)C] that lower cholesterol levels by inducing liver uptake of lipoproteins via galactose-recognizing hepatic receptors. In this study, we have investigated whether this strategy could also be applied to reduce elevated serum levels of the atherogenic lipoprotein(a) [Lp(a)]. Both TG(4A)C and TG(20A)C could be incorporated into Lp(a). Incorporation of these glycolipids induced a rapid clearance of Lp(a). Concomitantly, the hepatic uptake of 125I-Lp(a) was enhanced from 4 +/- 1% to 80 +/- 4% of the injected dose for TG(4A)C (P < .0001) and to 17 +/- 4% of the injected dose for TG(20A)C (P < .006). TG(4A)C was apparently more effective in accelerating the serum decay of 125I-Lp(a), which may be caused by the higher hydrophobicity of this glycolipid relative to TG(20A)C. The TG(4A)C- and TG(20A)C-induced stimulation of the serum decay and liver uptake of 125I-Lp(a) could be significantly inhibited (> 85%) by preinjection of N-acetyl-galactosamine (150 mg), indicating that galactose-recognizing receptors are involved in the liver uptake of the glycolipid/Lp(a) complexes. The TG(4A)C-induced liver uptake of 125I-Lp(a) could be ascribed mainly to Kupffer cells (76 +/- 7%), whereas the parenchymal liver cell was the major site for liver uptake of TG(20A)C-laden 125I-Lp(a) (55 +/- 12%). In conclusion, both TG(4A)C and TG(20A)C stimulate the catabolism of 125I-Lp(a) by enhancing hepatic uptake. Because endocytosis of the substrate via galactose-recognizing receptors on Kupffer and parenchymal liver cells is followed by lysosomal degradation, we anticipate that both approaches for Lp(a) targeting may prove valuable as therapeutic modalities for lowering atherogenic levels of Lp(a).